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∆N33 and ∆C68 deletion mutants were obtained by PCR truncation using specific primers on either the 5h or the. 3h end of NSP5 cDNA. For ∆N33, isoleucine at ...
Journal of General Virology (1998), 79, 2679–2686. Printed in Great Britain ...................................................................................................................................................................................................................................................................................

Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2 Ivka Afrikanova, Elsa Fabbretti, Maria C. Miozzo and Oscar R. Burrone International Centre for Genetic Engineering and Biotechnology, ICGEB, Area Science Park, Padriciano 99, 34012 Trieste, Italy

We have previously shown that a number of isoforms of the non-structural rotavirus protein NSP5 are found in virus-infected cells. These isoforms differ in their level of phosphorylation which, at least in part, appears to occur through autophosphorylation. NSP5 co-localizes with another non-structural protein, NSP2, in the viroplasms of infected cells where virus replication takes place. We now show that NSP5 can be chemically cross-linked in living cells with the viral polymerase VP1 and NSP2. Interaction of NSP5 with NSP2 was also demonstrated by coimmunoprecipitation of NSP2 and NSP5 from extracts of UV-treated rotavirus-infected cells. In

Introduction Rotaviruses are important pathogens that belong to the family Reoviridae, causing diarrhoea in several species. Infective particles replicate in the cytoplasm of infected cells. Although several reports have described the characterization of rotavirus replication intermediates (Gallegos & Patton, 1989 ; Patton & Gallegos, 1988), molecular details of the replication mechanism remain unclear. Partially purified intermediate complexes (or sub-viral particles) obtained from SA11-infected cells were able to catalyse run-off synthesis of endogenous dsRNAs (Patton, 1986), as well as complete replication of exogenously added viral ss (­)-RNAs (Chen et al., 1994 ; Wentz et al., 1996 ; Patton et al., 1996 ; E. Fabbretti & O. R. Burrone, unpublished). These results showed that the viral RNA polymerase structural proteins VP1, VP2 and VP3 are essential components of the replicase particles (Patton et al., 1997 ; Prasad et al., 1996). VP2 was demonstrated to be an essential element for the replicase– transcriptase activity that appears to be involved in the organization of the viral genome (Lawton et al., 1997). In virusinfected cells replication appears to take place within the viroplasms, where two other non-structural rotavirus proteins, Author for correspondence : Oscar Burrone. Fax ­39 040 226555. e-mail burrone!icgeb.trieste.it

0001-5561 # 1998 SGM

addition, in transient transfection assays, NSP5 phosphorylation in vivo was enhanced by coexpression of NSP2. An NSP5 C-terminal domain deletion mutant, was completely unable to be phosphorylated either in the presence or absence of NSP2. However, a 33 aa N-terminal deletion mutant of NSP5 was shown to become hyperphosphorylated in vivo and to be insensitive to NSP2 activation, suggesting a regulatory role for this domain in NSP5 phosphorylation and making it a candidate for the interaction with NSP2. These mutants also allow a preliminary mapping of NSP5 autophosphorylation activity.

NSP2 and NSP5, are localized (Petrie et al., 1982, 1984 ; Welch et al., 1989). These discrete structures are found in the cytoplasm at 2–3 h post-infection (p.i.). The NSP2 protein of 317 aa is encoded in gene segment 8 (in strain SA11) and possesses dsRNA and ssRNA binding capacity (Patton et al., 1993 ; Kattoura et al., 1992). Strains expressing temperature-sensitive mutants of this protein lose the ability to synthesize dsRNA and show a significant reduction in the synthesis of ssRNA (Chen et al., 1990). It has been shown that NSP2 is able to assemble in multimers and interact with the viral polymerase, VP1 (Kattoura et al., 1994). NSP5 is encoded in the longer ORF of genomic segment 11. It is a glycosylated phosphoprotein of 198 aa (in SA11) (Welch et al., 1989) with a high degree of conservation among different strains of group A rotaviruses including viruses with rearrangements of genome segment 11 (Gianbiagi et al., 1994). The NSP5 primary sequence is characterized by an overall high content (24 %) of serine and threonine. This is even higher (30±5 %) in the 131-aa-long N-terminal portion which is followed by a highly charged basic-acid-basic (B-A-B) domain (60 % charged residues) of 49 aa located between residues 131 and 179. The last 18 C-terminal aa are highly conserved even in group C viruses (Mattion et al., 1991). We have recently shown that NSP5 is hyperphosphorylated in vivo through a complex process, involving autophosphorylation, which gives

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rise to a number of different isoforms (26, 28 and 32–34 kDa) (Afrikanova et al., 1996). Similar results have also been obtained by other authors (Blackhall et al., 1997 ; Poncet et al., 1997). NSP5 is also characterized by having O-glycosidic monosaccharide residues of N-acetylglucosamine (NAcGlc) (Gonza! les & Burrone, 1991), a modification that involves serine residues in several proteins of cytoplasmic and nuclear localization (for review see Haltiwanger et al., 1992). Although NSP5 is found associated to cytoplasmic viroplasms from the early stages of infection (2 h p.i.), no function has yet been assigned to it. In order to investigate NSP5 interactions with other proteins we performed in vivo protein cross-linking of virus-infected cells and analysed NSP5 immunoprecipitates. Here we show that NSP5 interacts with two other rotaviral proteins, NSP2 and VP1, and that NSP2 plays a key role in the hyperphosphorylation of NSP5.

Methods + Cells, viruses and antibodies. MA104 cells were grown as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % foetal calf serum, 2 mM -glutamine, 50 mg}ml gentamycin (Gibco). Rotavirus simian SA11 strain was propagated and grown in MA104 cells as described (Estes et al., 1979). Guinea pig anti-NSP5 serum was produced as described (Gonza! les & Burrone, 1991). Mouse anti-NSP2 serum was obtained by the DNA-immunization technique (Raz et al., 1994). The complete NSP2 coding region was cloned in-frame downstream of a signal secretion leader peptide sequence in pUT-SEC plasmid (Li et al., 1997) and sequentially subcloned in pcDNA3 vector (Invitrogen) to generate pcDNA-spNSP2. Mice were injected intradermally twice with 100 µg of plasmid DNA. The serum was checked for specificity by immunofluorescence and Western blotting analysis against extracts of infected cells. + Radiolabelling with [35S]methionine, immunoprecipitation and PAGE analysis. Labelling of cells with [$&S]methionine was performed essentially as described by Afrikanova et al. (1996). Total cellular extracts were prepared in TNN buffer (100 mM Tris–HCl, pH 8±0, 250 mM NaCl, 0±5 % NP40, 1 mM PMSF) and used for immunoprecipitation and PAGE analysis as described (Afrikanova et al., 1996). + Chemical DSP-cross-linking. Cell monolayers were washed twice with PBS containing 1 mM Mg#+ and 1 mM Ca#+, overlaid with 1±5 ml washing buffer and dithiobis(succinimidylpropionate) (DSP) chemical cross-linker (Pierce) was added to the cells at a final concentration of 600 µM. The cells were then incubated at 4 °C for 15 min when the medium was removed. After washing with PBS the cross-linker was quenched twice with 1±5 ml 50 mM Tris pH 7±5, 150 mM NaCl buffer for 5 min at 4 °C. Cellular extracts were prepared in 100 µl TNN. + UV treatment of living cells. For UV treatment of cell monolayers, the medium was removed and the cells were washed twice with PBS, overlaid with 1 ml TBS buffer and exposed to UV-Stratalinker 1800 for 3 min (total dose 486 mJ) on ice. The distance between the cell monolayer and the UV lamp was kept at 15 cm. The extracts were prepared in RIPA buffer. Extracts were spun at 10 000 g for 7 min and supernatants used for immunoprecipitation analysis.

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+ In vitro phosphorylation. In vitro phosphorylation was carried out essentially as described by Afrikanova et al. (1996) by incubating Protein A–Sepharose beads (Pharmacia) containing immunoprecipitates with [γ-$#P]ATP (Amersham) for 30 min at 37 °C in kinase buffer (50 mM Tris–HCl, pH 8±0, 1±5 mM spermidine, 5 mM MgCl , 1 mM DTT, 5 % # glycerol). Phosphatase treatments were performed as described (Afrikanova et al., 1996). The immunoprecipitates were eluted by 4¬ Laemmli’s sample buffer at 96 °C for 3 min. The supernatants were analysed on 12 % PAGE. + Plasmid constructs. ∆N33 and ∆C68 deletion mutants were obtained by PCR truncation using specific primers on either the 5« or the 3« end of NSP5 cDNA. For ∆N33, isoleucine at position 34 was mutated to methionine. For ∆C68, a stop codon was introduced immediately downstream of Thr-130. NSP5 and mutated cDNA fragments were subcloned in pcDNA3 vector (Invitrogen) under the T7 promotor to generate pT7v-NSP5, pT7v-∆N33 and pT7v-∆C68. The NSP2 complete coding region was obtained by RT–PCR using total RNA preparation from SA11-infected cells and transferred to pcDNA3 (pT7v-NSP2). All amplified fragments of NSP5 mutants and NSP2 were cloned into pUC18 and sequenced with pUC forward and reverse primers before transferring to the pcDNA3 vector. + Transient transfections of MA104 cells. Confluent monolayers of MA104 cells in 30 mm diameter Petri dishes were infected with vaccinia virus (vTF7.3) at a multiplicity of 20 p.f.u. per cell (Fuerst et al., 1986). After 1 h, cells were transfected using the Transfectam reagent (Promega) as described by the manufacturer. We used 5 µg of total plasmid DNA (Qiagen purified) ; 3 µg corresponded to pT7v-NSP5 (or mutants pT7v-∆N33 and pT7v-∆C68) and 2 µg to either pT7v vector (in single transfections) or pT7v-NSP2 (in co-transfections). In all cases in which NSP2 was cotransfected with either wtNSP5 or mutants, the expression of NSP2 was assessed by immunofluorescence with the antiNSP2 antibody. The Transfectam reagent–DNA complexes were overlaid onto the cells and incubated for 12 to 15 h at 37 °C in a CO # incubator. Cells were then washed twice with PBS and cellular extracts prepared in 50 µl TNN lysis buffer at 4 °C. Extracts were spun at 10 000 g for 7 min and supernatants used in Western immunoblot analysis. + Immunoprecipitation and Western immunoblot analysis. Immunoprecipitation experiments were performed as described by Afrikanova et al. (1996). For immunoblots, lysates of transfected or cotransfected cells (corresponding to 0±5¬10& cells) were resolved on 12 % SDS–polyacrylamide gels (Laemmli et al., 1970), transferred onto a PVDF (Millipore) membrane and reacted to a guinea pig anti-NSP5 serum. An anti-guinea pig HRP-conjugated antibody (DAKO) was used as a second antibody, and developed by ECL-chemoluminescence (Amersham).

Results In vivo chemical cross-linking

In order to investigate possible interactions of NSP5 with other cellular or viral components, we performed in vivo chemical cross-linking of virus-infected cells, followed by immunoprecipitation with an anti-NSP5 specific antibody. We used DSP as a chemical cross-linker since it permeates into living cells, is able to cross-link proteins interacting at distances of up to 12 AI , and is sensitive to reducing agents. Analysis of the immunoprecipitates derived from cross-linked virusinfected cell extracts showed two new proteins in addition to

Rotavirus NSP2 up-regulates NSP5 phosphorylation

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Fig. 1. Analysis of DSP-cross-linked extracts. MA104 cells were mock infected or infected with rotavirus SA11, [35S]methionine-labelled and DSP-cross-linked where indicated. (a) Cellular extracts were immunoprecipitated with anti-NSP5 and analysed in reducing SDS–PAGE. (b) Immunoprecipitates from cross-linked extracts from virus-infected cells analysed by SDS–PAGE in non-reducing (lane 1) and reducing (lane 2) conditions ; lane 3 shows a total extract of virus-infected cells. (c) Immunoprecipitates from cross-linked or non-cross-linked extracts were treated with λ-Ppase before SDS–PAGE analysis. As a control immunoprecipitation with anti-NSP2 from virus-infected cell extracts is also shown (lane 5).

the different phosphorylated isoforms of NSP5 (Afrikanova et al., 1996). These two proteins migrated on SDS–PAGE identically to the viral proteins VP1 and NSP2 of molecular masses 120 and 35 kDa, respectively (Fig. 1 a, lane 4 ; 1 b , lanes 2–3). Other bands migrating like the two abundant viral proteins VP2 and VP6 were occasionally present in smaller amounts in the immunoprecipitates, but their presence was cross-linking-independent and varied according to the extent of washing of the immunoprecipitates. When then analysed by non-reducing SDS–PAGE, NSP5 immunoprecipitates consist of a complex of high molecular mass, with most of the material remaining in the top of the gel, and some free NSP2 and NSP5 (Fig. 1 b, lane 1). After reduction of the same immunoprecipitates, the three components become apparent : VP1, NSP2 and NSP5. A small proportion of both NSP2 and NSP5 was recovered from the immunoprecipitates even before treating with reducing agents (Fig. 1 b, lane 1). While this indicates that part of NSP5 is not involved in interactions with other proteins, for NSP2 it suggests that this protein is less efficiently cross-linked to other protein(s) of the complex. Treatment of the complex with λ-phosphatase (λPpase) before reduction and SDS–PAGE analysis produces the 26 kDa band of dephosphorylated NSP5 (Afrikanova et al., 1996), while the two other components of 35 and 120 kDa remain unchanged (Fig. 1 c). This result confirms that all the bands smaller than 35 kDa corresponded to the different phosphorylated isoforms of NSP5. Sequential immunoprecipitation with anti-NSP2 and antiNSP5 antibodies of cross-linked extracts indicated that most of the NSP2 interacting with NSP5 seems to remain buried within

the cross-linked complex not accessible to the precipitating antibody (data not shown). In vivo UV-treatment induces co-precipitation of NSP2 and NSP5

Exposure of virus-infected live cells to UV light was also used as an alternative approach to detect NSP5 interactions. The UV-cross-linking technique is mainly used to detect protein–nucleic acids interactions in vivo (Budowsky & Abdurashidova, 1989). However, as seen in Fig. 2, UV treatment of rotavirus-infected cells induced co-immunoprecipitation of NSP2 and NSP5, regardless of whether the cell extracts were prepared immediately after the [$&S]methioninelabelling pulse or following the 2 h chase. As expected a higher proportion of the 28 kDa NSP5 isoform was obtained after the chase period (Fig. 2 a). In the experiment shown in Fig. 2 (b), done with extract of cells labelled for 30 min, the isoforms of molecular masses higher than 28 kDa appeared as two sharper bands of 34 and 30 kDa in the immunoprecipitates from crosslinked cells (lane 1) when compared to those from non-crosslinked cells (lane 3). These two bands probably represent more homogeneously phosphorylated forms, since λ-Ppase treatment converted them all to the 26 kDa band, without affecting the migration of NSP2. Interestingly, while NSP2 clearly coprecipitated with anti-NSP5, no VP1 did so under these conditions. This result is particularly intriguing since NSP2 has been shown (by in vivo UV-cross-linking) to have ss- and dsRNA binding capacity. The co-precipitation of NSP2 could be the consequence of conformational modifications induced

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(b)

Fig. 2. SDS–PAGE analysis of anti-NSP5 immunoprecipitates from [35S]methionine-labelled virus-infected cells that were treated or not treated with UV light. (a) and (b) represent two different experiments. λ-Ppase treatment of the immunoprecipitate is indicated. The positions of NSP2 and NSP5 are indicated.

by UV treatment which increase its affinity for NSP5, so that a non-covalent interaction would be sufficiently stable to resist immunoprecipitate washings. Analysis of the immunoprecipitates under non-reducing SDS–PAGE yielded both NSP2 and NSP5, thus confirming the non-covalent nature of the interaction (not shown). However, we also considered the alternative possibility that UV-induced co-precipitation of NSP2 and NSP5 was the consequence of cross-linking of both proteins to a common RNA molecule. This hypothesis was quite unlikely since RNase digestion of UV-treated extracts before immunoprecipitation did not show any difference in the amount of NSP2 co-precipitated by anti-NSP5 antibodies (data not shown). In vitro phosphorylation of cross-linked NSP5

We have previously shown that NSP5 immunoprecipitated from virus-infected cells can be phosphorylated in vitro by incubation with [γ-$#P]ATP. When NSP5 immunoprecipitates obtained from extracts of [$&S]methionine-labelled and DSPcross-linked virus-infected cells were subjected to the in vitro phosphorylation assay, a substantial increase in the 32–34 kDa

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Fig. 3. In vitro phosphorylation assay of NSP5 from DSP-cross-linked extracts. Immunoprecipitates of [35S]methionine-labelled NSP5 from noncross-linked and DSP-cross-linked extracts were subjected to in vitro phosphorylation with [γ-32P]ATP and analysed by reducing SDS–PAGE. (a) and (b) show, for the same gel, autoradiograms corresponding to 35 S­32P and 32P only, respectively. λ-Ppase treatment of the immunoprecipitates is indicated.

hyperphosphorylated-form bands was obtained, compared with the non-cross-linked cells (Fig. 3 a, b, lanes 1 and 3). Since we and others have shown that NSP5 is itself a kinase with autophosphorylation activity (Afrikanova et al., 1996 ; Blackhall et al., 1997 ; Poncet et al., 1997), the results shown suggest that the association of NSP5 with NSP2, VP1 or both has an activating effect on the kinase activity of NSP5 or on NSP5 as the substrate of phosphorylation. While the immunoprecipitates obtained from the cross-linked extracts represented a complex (or complexes) of three proteins, VP1, NSP2 and NSP5, neither VP1 nor NSP2 appeared to be phosphorylated in the in vitro assay. As shown in Fig. 3 (b), lanes 2 and 4, λ-Ppase treatment of the phosphorylated samples removed almost all the phosphate. The total amount of NSP5 in the immunoprecipitates subjected to the in vitro phosphorylation assay was the same in cross-linked and noncross-linked extracts, as evidenced by the λ-Ppase treatment (Fig. 3 a, lanes 2 and 4). It is noteworthy to mention that we have also confirmed that neither VP1 nor NSP2 is phosphorylated in vivo (data not shown). NSP2 activates phosphorylation of NSP5 in vivo

The possibility of NSP2 alone being sufficient for the activation of NSP5 hyperphosphorylation was directly tested in vivo, in experiments in which NSP5 and NSP2 were coexpressed in the absence of any other rotaviral protein. We used the T7–vaccinia virus expression system, co-transfecting plasmids containing the complete coding region of NSP5 and NSP2 (pT7v-NSP5 and pT7v-NSP2) under the control of the T7 polymerase promoter into MA104 cells. Total extracts of transfected cells previously infected with T7–vaccinia were

Rotavirus NSP2 up-regulates NSP5 phosphorylation

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Fig. 4. Anti-NSP5 Western immunoblot of cellular extracts of MA104 cells transfected with pT7v-NSP5 or co-transfected with pT7v-NSP5 and pT7vNSP2, as indicated. (a) and (b) show two independent experiments. Where indicated, λ-Ppase treatment of the extract was performed before PAGE. Open and closed arrowheads indicate the NSP5 26 kDa precursor and phosphorylated forms, respectively.

analysed by Western immunoblotting to visualize the different NSP5 isoforms. Fig. 4 shows the results of two independent experiments. Expression of NSP5 alone gives rise to the 26 kDa and, to a lesser extent, 28 kDa bands and no other NSP5 product. This is in complete agreement with our previous observations on NSP5 phosphorylation in MA104 stable transfectants (Afrikanova et al., 1996). On the other hand, when both NSP2 and NSP5 were co-expressed, a clearly increased phosphorylation (28 kDa band) and hyperphosphorylation (32–34 kDa bands) of NSP5 were obtained. As expected all these bands were sensitive to λ-Ppase. This result demonstrated that NSP5 hyperphosphorylation is activated in vivo by NSP2, and suggested a direct interaction of NSP2 and NSP5 in the absence of any other viral protein. It remains to be determined whether the up-regulation of phosphorylation is the consequence of an increased kinase activity of NSP5 or NSP5 becoming a better substrate. A regulatory role of the NSP5 N-terminal domain on phosphorylation

Two deletion mutants of NSP5 were constructed, lacking, respectively, the 33 aa of the N-terminal portion (∆N33) or the 68 aa of the C-terminal domain (∆C68) (Fig. 5 a). In the case of ∆N33, Ile-34 was changed to Met. The resulting protein contains 165 aa with an apparent molecular mass of 22 kDa upon reducing PAGE of the in vitro translated product. The ∆C68 mutant of 130 aa (apparent molecular mass 18 kDa), lacks the B-A-B domain (aa 130–179) and the highly conserved terminal 20 aa. When ∆N33 was expressed in vivo by transfection using the T7–vaccinia system, a strong phosphorylation was observed with the appearance of several bands of apparent molecular mass higher than 22 kDa (filled arrowheads in Fig. 5). All these bands corresponded to hyperphosphorylated forms, as revealed by their sensitivity to λ-Ppase (Fig. 5 d).

Fig. 5. In vivo phosphorylation of NSP5 and NSP5 deletion mutants. (a) Schematic representation of wild-type NSP5 and NSP5 deletion mutants ∆N33 and ∆C68. The relative content of Ser­Thr residues of the different NSP5 regions is shown. B-A-B indicates the acid-basic-acid domain. (b)–(d) NSP5 Western immunoblot of cell extracts transfected with pT7v-NSP5, pT7v-∆N33 and pT7v-∆C68 (b) and, where indicated, co-transfected with pT7v-NSP2 (c). The extract from pT7v-∆N33 transfected cells was treated with λ-Ppase before PAGE (d). Open arrowheads indicate the NSP5 wild-type (26 kDa), ∆N33 (22 kDa) and ∆C68 (18 kDa) precursors and closed arrowheads their corresponding phosphorylated forms.

These data suggest that the lack of the 33 aa N-terminal domain allows NSP5 to become hyperphosphorylated. Interestingly, co-expression of NSP2 and ∆N33 did not produce any further activation of phosphorylation (Fig. 5 c). On the other hand, the ∆C68 mutant was not phosphorylated at all, even when co-expressed with NSP2. Taken together, these results indicate that the N-terminal 33 aa domain plays a regulatory role in the phosphorylation of NSP5, by reducing the ability of the complete molecule to become hyperphosphorylated when expressed in vivo in the absence of other viral proteins. This domain probably also regulates the proportion of higher molecular mass isoforms in infected cells.

Discussion We have previously shown that the rotavirus non-structural protein NSP5 is hyperphosphorylated during the virus replicative-cycle. As a consequence, a heterogeneous set of hyperphosphorylated forms with molecular masses of up to 32–34 kDa are produced in addition to the main isoforms of 26 and 28 kDa. NSP5 appears to have autophosphorylation

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activity in the absence of any other viral or cellular protein (Afrikanova et al., 1996 ; Blackhall et al., 1997 ; Poncet et al., 1997). The 26 kDa protein, expressed in E. coli and purified, was autophosphorylated in an in vitro kinase assay (Blackhall et al., 1997 ; Poncet et al., 1997). However, we have shown that NSP5 obtained by in vitro translation, becomes phosphorylated and transformed into the 28 kDa form only when extracts from infected cells were added to the assay. Moreover, hyperphosphorylation of the 32–34 kDa forms in vitro can be obtained using NSP5 protein derived from virus-infected cells (Afrikanova et al., 1996). These results supported the idea that phosphorylation of NSP5 is a complex process that initiates with the transformation of the 26 kDa precursor into the 28 kDa form which, in virus-infected cells, is hyperphosphorylated giving rise to the 32–34 kDa isoforms. This suggests that control of NSP5 phosphorylation may be regulated through interactions with other proteins. We have now demonstrated that NSP5 hyperphosphorylation can also take place in vivo when NSP5 is coexpressed with NSP2. In addition, we demonstrated, by crosslinking and immunoprecipitation, a direct interaction of NSP5 with NSP2 and the viral polymerase, VP1. NSP2 is a rotaviral non-structural protein which has been previously found to interact also with the viral polymerase and to form homomultimers (Kattoura et al., 1994). NSP2 binds to viral ssRNAs and can be cross-linked in vivo to the 11 dsRNA genomic segments. The 10S homomultimers possess ssRNA binding activity as well. Therefore, it is possible that the interaction(s) between NSP5 and NSP2 is facilitated when the latter is bound to RNA, as suggested by the UV-treatment experiments. It has been demonstrated that under the same conditions used in our experiments, UV induces cross-linking of NSP2 to RNA (Kattoura et al., 1992). It is therefore possible that following UV treatment, NSP2 may acquire a conformation that favours and stabilizes a non-covalent interaction with NSP5. This association does not involve disulphide bonds since the two proteins separated in PAGE under non-reducing conditions (not shown). An interesting observation is that the viral polymerase, VP1, co-precipitates with NSP5 from DSP-cross-linked extracts, suggesting that it could also be an additional component of NSP2–NSP5 complexes. However, we obtained different results regarding the presence of VP1 in immunoprecipitates from DSP-cross-linked or UV-treated extracts. The fact that VP1 does not co-precipitate from UV-treated extracts suggests that the nature of the NSP5–NSP2 and NSP5–VP1 interactions is different. The NSP2–NSP5 interaction appears to have functional consequences regarding the level of phosphorylation of NSP5. A first indication of this fact was obtained in the in vitro phosphorylation assays of DSP-cross-linked extracts, which was later confirmed by co-expression of both proteins in vivo. In the latter case, the NSP5-phosphorylation pattern obtained was very similar to that observed in infected cells. These

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results demonstrated that NSP2 up-regulates the phosphorylation of NSP5 in vivo. Even though we cannot rule out a role for cellular kinases in NSP5 phosphorylation, we favour the idea that a direct interaction between NSP2 and NSP5 is essential for NSP5 hyperphosphorylation. Many kinases become activated by phosphorylation by other kinases (Superti-Furga & Courtneidge, 1995 ; Cooper & Howell, 1993). In the present case, NSP5 hyperphosphorylation could be the consequence of an indirect effect of NSP2 on cellular kinases. However, our results support the idea of a direct interaction because : (a) NSP2 co-immunoprecipitated with NSP5 following chemical cross-linking and UV treatment of infected cells ; (b) NSP2 enhanced the hyperphosphorylation of NSP5 32–34 kDa bands in in vitro phosphorylation experiments ; and (c) co-expression of NSP2 and NSP5 induced the formation of the 32–34 kDa phosphorylated isoforms in cells that would have otherwise expressed only the 26 and 28 kDa forms. The results obtained with the ∆N33 and ∆C68 deletion mutants of NSP5 also support the hypothesis of an interaction with NSP2. While deletion of the N-terminal portion of NSP5 contributed to an enormous increase in phosphorylation as compared to the wild-type NSP5, deletion of the C-terminal domain completely abolished NSP5 phosphorylation, even in the presence of NSP2. This reinforces the hypothesis that NSP5 is autophosphorylated in vivo, since ∆C68 lacks the highly conserved 178–198 region, with homology to guanido kinases (Mattion et al., 1994), but contains the most serine- and threonine-rich portion of the molecule. The inability of NSP2 to activate phosphorylation of ∆N33 suggested that this domain could be important for the NSP2–NSP5 interaction. We hypothesize that the N-terminal portion of NSP5 plays the role of an inhibitory domain of NSP5 hyperphosphorylation. Up-regulation of this activity could be induced by conformational changes resulting from the interaction of NSP5 with NSP2. Such interaction has been also reported using a yeast two-hybrid system (Poncet et al., 1997). In addition, the results with the ∆N33 and ∆C68 mutants suggest that the putative kinase activity maps to the Cterminal region of NSP5. Interestingly, although the 33 aa Nterminal domain has the highest content of Ser and Thr (48 %), its absence does not preclude NSP5 from becoming hyperphosphorylated, indicating that the sites of such phosphorylation reside in other regions of the protein. The three proteins that we found to interact (NSP2, NSP5 and VP1) are present in the viroplasms of infected cells. NSP2 has been reported to interact with VP1 (Kattoura et al., 1994). Recently, using a monoclonal anti-NSP2 antibody, a viral RNA–protein complex with replicase activity was recovered (Aponte et al., 1996). This complex contained the structural proteins VP1, VP2, VP6, and NSP2, and was able to complete nascent RNA negative-strands, but did not use exogenous RNAs as templates (Patton, 1996). Other authors have shown

Rotavirus NSP2 up-regulates NSP5 phosphorylation

that, in vitro, replicative complexes able to uptake exogenously added viral ss (­)-RNAs contain, among other viral proteins, NSP2 and NSP5 (Patton et al., 1996 ; E. Fabbretti & O. R. Burrone, unpublished). Numerous phosphoproteins have been implicated in the replication of other RNA viruses as well (Kapoor et al., 1995 ; Kann et al., 1995 ; Chang et al., 1994 ; Lastarza et al., 1994). Our finding that NSP5 consists of a wide range of phosphorylated isoforms, with phosphorylation being up-regulated by NSP2, suggests a possible involvement of NSP5 in the process of sorting and packaging of the viral RNA during the replicative cycle. We are grateful to Mauro Sturnega for excellent technical assistance, Gulnara Abdurashidova for helpful discussions and Rodolfo Garcia for discussions and critical reading of the manuscript.

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